Feeding behavior is critical for animal survival, and is also a fundamental aspect of energy homeostasis. The growing incidence of eating disorders and their associated health costs have led to intensive research efforts directed to understand the mechanisms and signaling pathways that control and regulate food intake and energy homeostasis. This process appears to be regulated by a highly complex neuroendocrine system involving a multitude of neuropeptides. However, given the high chemical complexity and wide distribution of neuropeptides, the precise molecular mechanisms at the cellular and network levels remain elusive. This is, in large part, due to a lack of analytical capabilities to measure and identify these low abundance endogenous signaling molecules in a complex microenvironment. Clearly, the development of highly sensitive and selective analytical tools for neuropeptide identification and quantitation is in great demand. This project aims to fill this gap by constructing and implementing a unique analytical measurement platform and developing improved mass spectrometry (MS) - based methodologies for probing peptidergic signaling in feeding with enhanced sensitivity and selectivity. We have chosen to study the simpler and well-defined crustacean stomatogastric nervous system (STNS) and its associated neuroendocrine organs, to facilitate the technology development and validation. Furthermore, the wealth of information about the neuropeptides present in this model system and its well-defined physiology provide unique opportunities to address fundamental neuroscience problems related to the neuropeptidergic modulation of complex behaviors such as feeding. The specific aims of this project include: (1) To develop direct tissue in situ peptide profiling and quantitation via in-cell combination (QUICC) methodologies by matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS). Major neuroendocrine organs isolated from food deprived and satiated animals will be analyzed and compared for their peptide content; (2) To develop in vivo microdialysis sampling techniques and isotopic labeling strategies coupled to nanoflow LC/MS for differential display of circulating peptides in response to feeding; (3) To develop a hybrid strategy combining the use of isotope-assisted de novo MS/MS sequencing and sequence homology searching to identify and discover novel neuropeptides, with focus on the peptides showing differential expression and secretion in response to feeding; (4) To test physiological effects of the newly discovered peptides on the feeding circuits (gastric mill and pyloric neuronal networks in the stomatogastric ganglion). Collectively, these proposed experiments will develop and illustrate improved methods and capabilities for neuropeptide analysis. This project will also discover a large number of new peptides and provide the neurochemical basis toward a detailed mechanistic understanding of the peptidergic regulation of feeding behavior. The molecular insights gained from studying such a small system can be transferred to the larger, more complex vertebrate systems and could potentially lead to the development of new therapeutic strategies for feeding disorders. [unreadable] [unreadable] [unreadable]